1. Field of the Invention
This invention relates to a nucleic acid encoding a multi-functional antimicrobial fusion protein formed from a nucleic acid encoding a functional module or domain of a particular peptidoglycan hydrolase, e.g., an endolysin which specifically attacks the peptidoglycan cell wall of a bacteria of one genus, specie or substrain in combination with additional nucleic acids encoding functional modules or domains of other peptidoglycan hydrolases which specifically attack the peptidoglycan cell walls of additional bacteria from different genus, specie or substrains. A preferred embodiment relates to an isolated bifunctional peptidoglycan hydrolase fusion protein, wherein one module of the fusion endolysin is specific for a specific bond in the peptidoglycan wall of Staphylococcus aureus and the second module is specific for a specific bond in the peptidoglycan wall of Streptococcus spp. (agalactiae, dysgalactia and, uberis). The invention further relates to methods of treating diseases caused by the bacteria for which the individual modules of the fusion protein are specific.
2. Description of the Relevant Art
Novel antimicrobials are desperately needed to stem the tide of increasing incidence of antimicrobial resistance. The social and economic impact of such resistance has been summarized and characterized as “accumulating and accelerating, while the world's tools for combating it decrease in power and number: More than 90 percent of Staphylococcus aureus strains are resistant to penicillin and other related antibiotics. There is an alarming rise in the incidence of enterococci (the streptococcus that is the most common cause of hospital-acquired infections) resistant to the antibiotic vancomycin, often the last weapon for defeating these pathogens. As many as 40 percent of strains of pneumococci in some parts of the United States are now partly or completely resistant to penicillin and a number of antibiotics. The cost of these dynamics, especially multidrug resistance, is also rising—in terms of mortality, disability, and dollars. Antibiotic-resistant bacteria generate a minimum of $4 billion to $5 billion in costs to U.S. society and individuals yearly, and in 1992, the 19,000 deaths directly caused by hospital-acquired infections made them the eleventh leading cause of death in the U.S. population.” (1998. Workshop Summary of the Forum on Emerging Infections, In: Antimicrobial Resistance: Issues and Options, Harrison and Lederberg, eds., Institute of Medicine, National Academy Press, Washington, D.C., page 1.)
Antibiotic-resistant bacteria are also of concern with regard to animal diseases. In the USA alone, losses due to mastitis are estimated to be between $1.7 billion (Bramley et al. 1996. In: Current Concepts of Bovine Mastitis, 4th Edition, National Mastitis Council, Inc., Madison, Wis.) and $2 billion annually (Sordillo et a/2002. J. Mammary Gland Biol. Neoplasia 7(2): 135-146). In a recent regional survey of mastitis in the Pennsylvania and New York, Staphylococcus aureus and Streptococcus agalactiae (Group B Streptococcus; GBS) were each shown to be responsible for up to 20% of the mastitis in this region (Wilson et at 1997. J. Dairy Sci. 80(10): 2592-2598). Streptococcus dysgalactiae (Group C Streptococcus; GCS) and Streptococcus uberis are also serious mastitis-causing pathogens in the USA, with S. uberis also being responsible for 20% of the mastitis in the UK (Veterinary Laboratories Agency & Scottish Agricultural College Survey).
These bacteria are causative agents for both animal and human diseases. S. aureus, GBS and GCS, are also known human pathogens with 90% of the isolated S. aureus strains showing drug resistance (see above). GBS is a major neonatal pathogen. Up to 20% of pregnant women are colonized vaginally with GBS that can result in potentially fatal neonatal infections (Regan et al. 1996. Am. J. Obstet. Gynecol. 174(4): 1354-1360). GCS has recently been shown to be a cause of acute pharyngitis in children (Zaoutis et al. 2004. Clin. Microbiol. Infect. 10(1): 37-40)
A recurring theme in the quest for new antimicrobials is the use of bacteriophage therapy. Bacteriophage are viruses that attack bacteria. Bacteriophage therapy has been in existence for nearly 100 years having reached its popularity in Eastern Europe in the early 1900s, but fell out of favor after the discovery of antibiotics in the mid 1900s (reviewed by Mathur et al. 2003. J. Assoc. Physicians India 51: 593-596). The phage infects a single cell, replicates itself within the host cell, and then explodes the host cell releasing the progeny phage which then go on to repeat the cycle. Each phage genome codes for lytic proteins that degrade the bacterial cell wall peptidoglycan and allow the phage to escape.
Peptidoglycans are a major structural component of both Gram positive and Gram negative bacterial cell walls. They are the major strength element of the bacterial cell wall, are up to 40 layers thick, and are composed of a complex molecule with a sugar backbone (alternating N-acetyl glucosamine and N-acetyl muramic acid residues). This backbone is cross linked with covalently attached peptides, allowing the bacteria to withstand the 3-4 atmospheres of osmotic pressure that exists across the bacterial cell wall, see FIG. 1 (Chatterjee et al. 1977. J. Theor. Biol. 68(3): 385-390). Although peptidoglycan structure is similar between species, with the oligosaccharide backbone present in all, there is also a great deal of cell wall diversity between different bacterial species.
Bacteriophage are very specific and usually limited in their host range, thus a single strain of phage will not infect or kill all bacteria. This specificity is in part derived from the specificity of their lytic genes (namely endolysins) for a specific covalent bond in the cell wall peptidoglycan. At this time, there are four major classes of enzymatic activities which have been identified in endolysins that can degrade peptidoglycan, 1) N-acetyl muramidase (lysozyme like), 2) N-acetylglucosaminidase, 3) N-acetylmuramyl-L-alanine amidase and 4) endopeptidase (FIG. 1).
Endolysins are modular in nature with each module harboring a specific enzymatic activity. These modular sequences are highly conserved between phages that have very different host ranges. It is also common to have multiple modules linked together to form a single endolysin, that can then attack the cell wall peptidoglycan at multiple sites in the molecule. A well characterized example is the Streptococcus pneumoniae phage Dp-1 endolysin which has a choline binding domain at the carboxyl terminus and an amidase module at the amino terminus (Garcia et al. 1988. Proc. Natl. Acad. Sci. U.S.A. 85: 914-918). Choline binding is necessary for full enzymatic activity. The choline-binding module may have been obtained through horizontal transfer from a Lactobacillus strain, while the amidase domain was likely obtained from a Streptococcus autolysin, lytA. The choline domain can be exchanged between various gram positive bacteria and their bacteriophage enzymes and still maintains its function (Lopez et al. 1997. Microb. Drug Resist 3(2): 199-211).
These mastitis-causing strains are killed by endolysins: Streptococcus agalactiae, S. dysgalactiae (Pritchard et al. 2004. Microbiology 150(7): 2079-2087) and S. uberis (personal communication, David Donovan), are killed by B30 endolysin; Staphylococcus aureus and some coagulase negative Staphylococcus, are killed by lysostaphin (Cisani et al. 1982. Antimicrob. Agents and Chemo. 21:531-535).
In the quest for antimicrobials against mastitis-causing bacteria, agents must be found that can target our pest organisms very specifically. This is not just to reduce the potential for resistance development, but also to prevent damage to commercially important organisms that are necessary for the downstream processing of milk into yogurt and cheese. The prevention of mastitis would not just benefit animal health, but also food quality. Any antimicrobial that is specific for a given pathogen will potentially reduce the use of broad range antibiotics and thus help prevent the onslaught of multi drug resistant varieties.
Thus, to counter the rise of drug resistant pathogenic bacteria, there is a need for new specific antimicrobial treatments. Reagents shown to be very specific for the genera, species or substrains of concern would give better effective control of economically important diseases and would therefore be ideal candidates for therapeutic treatments.